Flaviviruses populate the NIAID's Category A, B, and C lists of viruses. Vaccines are urgently needed for multiple flavivirus diseases, notably dengue and West Nile encephalitis. To develop more effective vaccines a comprehensive understanding of how flaviviruses trigger immune responses is needed. To address this need, we developed single-cycle flaviviruses and packaging cell systems that produce them at high titers. These single-cycle viruses include West Nile virus (WNV) -derived viral replicon particles (VRPs) and a WN encephalitis vaccine candidate named Replivax. Both particles encode self-replicating WNV RNA genomes (replicons) deficient in one or more genes required for genome packaging. VRPs and Replivax mimic many aspects of WNV infection (receptor binding, uptake, RNA release, and genome replication). However, VRPs and Replivax cannot produce progeny virions in normal cells, and hence are not pathogenic. Nevertheless, Replivax encodes a protective immunogen, the sub-viral particle (SVP), and has proven to be a remarkably potent vaccine candidate. VRPs can be modified to express marker genes (to allow tracking in vivo), further enhancing their utility. Our single-cycle particles target the draining lymph nodes of mice, where they infect macrophages and induce high levels of type I interferon (IFN), suggesting that IFN is critical for directing the adaptive immune response to our vaccine. However, mice deficient in type I IFN receptors produced antibody responses to Replivax indistinguishable from WT animals, suggesting that IFN was not linking innate and adaptive immunity. In the process of learning how WNV induces IFN, we examined how ex vivo cultures of murine bone marrow-derived dendritic cells responded to WNV VRP infection. These studies showed that protein kinase R (PKR) appeared to be acting as a pattern-recognition receptor (PRR) that triggered IFN synthesis. Further, VRP-inoculated PKR-deficient mice produced lower levels of IFN and lower levels of WNV-specific antibodies than WT mice, indicating that PRR pathways (PRRPs) control both innate and adaptive responses. Based on these studies we hypothesize that: Recognition of Replivax via PRRs results in the engagement of multiple PRRPs, which may act independently, or in concert, to induce innate and adaptive immunity. This hypothesis will be evaluated in 3 specific aims in which we will elucidate the effect of macrophage depletion and deficiencies in specific PRRPs on the 1) innate, 2) humoral, and 3) cellular immune response in mice inoculated with these unique single-cycle flavivirus particles.